Currently available wound healing therapies involve the administration of therapeutic proteins. Such therapeutic proteins may include regulatory factors involved in the normal healing process such as systemic hormones, cytokines, growth factors and other proteins that regulate proliferation and differentiation of cells. Growth factors, cytokines and hormones reported to have such wound healing capacity include, for example, the transforming growth factor-superfamily (TGF) of proteins (Cox, Cell Biol. Int. 19:357-371, 1995) acidic fibroblast growth factor (FGF) (Slavin, Cell Biol. Int. 19:431-444, 1995), macrophage-colony stimulating factor (M-CSF) and calcium regulatory agents such as parathyroid hormone (PTH).
A number of problems are associated with the use of therapeutic proteins, i.e., cytokines, in wound healing therapies. First, the purification and/or recombinant production of therapeutic proteins is often an expensive and time-consuming process. Despite best efforts, however, purified protein preparations are often unstable making storage and use cumbersome, and protein instability can lead to unexpected inflammatory reactions (to protein breakdown products) that are toxic to the host.
Second, systemic delivery of therapeutic proteins, i.e., cytokines, can be associated with serious unwanted side effects in unwounded tissue. Due to inefficient delivery to specific cells and tissues in the body, administration of high doses of protein are required to ensure that sufficient amounts of the protein reach the appropriate tissue target. Because of the short half life in the body due to proteolytic degradation, the proteins must also be administered repeatedly which may give rise to an immune reaction to the therapeutic proteins. The circulation of high doses of therapeutic proteins is often toxic due to pleiotropic effects of the administered protein, and may give rise to serious side effects.
Third, exogenous delivery of recombinant proteins is inefficient. Attempts have been made to limit the administration of high levels of protein through immobilization of therapeutic protein at the target site. However, this therapeutic approach complicates the readministration of the protein for repeated dosing.
Fourth, for a variety of proteins such as membrane receptors, transcription factors and intracellular binding proteins, biological activity is dependent on correct expression and localization in the cell. For many proteins, correct cellular localization occurs as the protein is post-translationally modified inside the cells. Therefore, such proteins cannot be administered exogenously in such a way as to be taken up and properly localized inside the cell.
As these problems attest, current recombinant protein therapies for wound healing are flawed, because they do not present a rational method for delivery of exogenous proteins. These proteins, e.g., cytokines, are normally produced at their site of action in physiological amounts and efficiently delivered to cell surface signaling receptors.
Gene Therapy
Gene therapy was originally conceived of as a specific gene replacement therapy for correction of heritable defects to deliver functionally active therapeutic genes into targeted cells. Initial efforts toward somatic gene therapy have relied on indirect means of introducing genes into tissues, called ex vivo gene therapy, e.g., target cells are removed from the body, transfected or infected with vectors carrying recombinant genes, and re-implanted into the body (“autologous cell transfer”). A variety of transfection techniques are currently available and used to transfer DNA in vitro into cells; including calcium phosphate-DNA precipitation, DEAE-Dextran transfection, electroporation, liposome mediated DNA transfer or transduction with recombinant viral vectors. Such ex vivo treatment protocols have been proposed to transfer DNA into a variety of different cell types including epithelial cells (U.S. Pat. No. 4,868,116; Morgan and Mulligan WO87/00201; Morgan et al., Science 237:1476-1479, 1987; Morgan and Mulligan, U.S. Pat. No. 4,980,286), endothelial cells (WO89/05345), hepatocytes (WO89/07136; Wolff et al., Proc. Natl. Acad. Sci. USA 84:3344-3348, 1987; Ledley et al., Proc. Natl. Acad. Sci. 84:5335-5339, 1987; Wilson and Mulligan, WO89/07136; Wilson et al., Proc. Natl. Acad. Sci. 87:8437-8441, 1990) fibroblasts (Palmer et al., Proc. Natl. Acad. Sci. USA 84:1055-1059, 1987; Anson et al., 1987, Mol. Biol. Med. 4:11-20; Rosenberg et al., Science 242:1575-1578, 1988; Naughton & Naughton, U.S. Pat. No. 4,963,489), lymphocytes (Anderson et al., U.S. Pat. No. 5,399,346; Blaese, R. M. et al., Science 270:475-480, 1995) and hematopoietic stem cells (Lim, B. et al., Proc. Natl. Acad. Sci. USA 86:8892-8896, 1989; Anderson et al., U.S. Pat. No. 5,399,346).
Direct in vivo gene transfer has recently been attempted with formulations of DNA trapped in liposomes (Ledley et al., J. Pediatrics 110:1, 1987); or in proteoliposomes that contain viral envelope receptor proteins (Nicolau et al., Proc. Natl. Acad. Sci. U.S.A. 80:1068, 1983); and DNA coupled to a polylysine-glycoprotein carrier complex. In addition, “gene guns” have been used for gene delivery into cells (Australian Patent No. 9068389). It has even been speculated that naked DNA, or DNA associated with liposomes, can be formulated in liquid carrier solutions for injection into interstitial spaces for transfer of DNA into cells (Felgner, WO90/11092).
Perhaps one of the greatest problems associated with currently devised gene therapies, whether ex vivo or in vivo, is the inability to transfer DNA efficiently into a targeted cell population and to achieve a useful level expression of the gene product in vivo. Viral vectors are regarded as the most efficient system, and recombinant replication-defective viral vectors have been used to transduce (i.e., infect) cells both ex vivo and in vivo. Such vectors have included retroviral, adenovirus and adeno-associated and herpes viral vectors. While highly efficient at gene transfer, the major disadvantages associated with the use of viral vectors include the inability of many viral vectors to infect non-dividing cells; problems associated with insertional mutagenesis; inflammatory reactions to the virus and potential helper virus production, and/or production and transmission of harmful virus to other human patients.
In addition to the low efficiency of most cell types to take up and express foreign DNA, many targeted cell populations are found in such low numbers in the body that the efficiency of presentation of DNA to the specific targeted cell types is even further diminished. At present, no protocol or method, currently exists to increase the efficiency with which DNA is targeted to the targeted cell population.
Accordingly, there is a need in the art for efficiently transferring nucleic acids into a targeted cell population and to achieve high level expression of the transferred nucleic acids in vivo.
Fluid Space
Cells and tissues of the body are composed of and surrounded by fluids. Body fluids include both intracellular and extracellular fluids. Intracellular fluids are body fluids that are within the cell membranes. Generally, intracellular fluids are composed of water and dissolved solutes. Extracellular fluids include body fluids outside of cells, such as interstitial fluid, plasma, lymph, cerebrospinal fluid, etc. Extracellular fluids consist of ultrafiltrates of the blood plasma and transcellular fluid that is produced by active cellular secretion. Extracellular fluids provide a constant external environment for cells. Interstitial fluid is a type of extracellular fluid that bathes the cells of most tissues but is not within the confines of the blood or lymph vessels and is not a transcellular fluid. Interstitial fluid is formed by filtration through the blood capillaries and is drained away as lymph. Examples of interstitial fluids include allantoic fluid, amniotic fluid, ascitic fluid, follicular fluid, pericardial fluid, seminal fluid, and synovial fluid.
Extracellular fluids generally accumulate in fluid spaces, which includes any space or cavity capable of containing fluid. It is not necessary for fluid spaces to actually contain fluid. Fluid spaces that do not contain fluid are referred to histologically as “potential spaces.” Examples of fluid spaces include follicles of the thyroid, joint cavities, tendon sheaths, the vitreous of the eye, the four ventricles of the brain, the subarachnoid space, the articular space, the inner and middle ear, the central canal of the spinal cord, the pericardium, the peritoneal cavity, pleural cavity, and retroperitoneal cavity. Blood vessels such as veins, arteries and capillaries are not considered fluid spaces.
Efforts to perform gene therapy on tissues associated with fluid spaces include the introduction of a gene therapy vector directly into a fluid space under conditions in which cells associated with the fluid space can incorporate the nucleic acid vector (Ledley and O'Malley, U.S. Pat. No. 5,792,751). These methods generally rely on the ability of target tissues directly in contact with a fluid space to take up introduced nucleic acids by pinocytosis, phagocytosis, receptor mediated uptake, or membrane fusion. In addition, these methods depend upon the ability of the transduced tissues to express the product of the introduced gene therapy vector. Accordingly, vectors capable of tissue specific expression are necessary to direct expression in defined tissues of interest. Another drawback of the methodology described by Ledley is that the introduction of DNA expression vectors directly into a fluid space requires diffusion to the site of treatment as well as requiring liquid formulations, which may require refrigeration and associated sterile techniques.